PCR product of unlinearized plasmid - (Feb/14/2008 )
ive conducted a laboratory practical where we transformed ampicillin resistance into E.coil using pUC19. as part of it, we placed a linear sample and an unlinear sample as templates into PCR, and loaded the products along with a restriction digest of the plasmid, and a whole undigested plasmid into an agarose gel for electrophoresis.
why would unlinearised plasmid produced a different product from PCR?
and my gel results have confused me my linearised plasmid PCR product and circular plasmid product have produced the same results, my restriction digest has two bands, and its migrated the same distance as an undigested whole plasmid, and the linearised plasmid has two very faint bands at the same place as the restriction digest
First of all, you might want to ask yourself what would you expect to see in each lane on your gel. For example, how big do you expect the DNA from your PCRs to be versus the size of the parent plasmid? Should a linear and a cut plasmid give the same results in your PCR or not? Why?
Furthermore, what is the expected difference between your 'uncut' plasmid, your 'linearised' plasmid and your 'restriction digested' plasmid? What size DNA fragments do you expect from each? Also, should the uncut, supercoiled plasmid electrophorese in a similar manner as the linear plasmid or not?
I'm sure if you think this through carefully you can answer most of your questions yourself.